Methods of increasing platelet and hematopoietic stem cell production

ABSTRACT

A method of increasing hematopoietic stem cell production is disclosed. The method includes administering a TPO mimetic compound to a subject. Pharmaceutical compositions including a TPO mimetic compound and a pharmaceutically acceptable carrier are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/667,096, filed Sep. 18, 2003, which in turn claims priority toApplication Nos. 60/411,779 and 60/411,700 filed on Sep. 18, 2002, andApplication No. 60/498,740, filed Aug. 28, 2003, the entire contents ofwhich are incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

Thrombopoietin (TPO), initially cloned as a major regulator of plateletproduction, plays a pivotal role in hematopoietic stem cell (HSC)biology. Kaushansky et al., Nature, 369:568-571 (1994). Virtually allprimitive HSC that display repopulating activity express c-Mpl, thereceptor of TPO. Solar et al., Blood, 92:4-10 (1998). TPO alone or incombination with other early acting cytokines, such as stem cell factor(SCF), interleukin 3 (IL-3), or Flt-3 ligand enhance proliferation ofprimative HSC in vitro. Ku et al., Blood, 87:4544-4551 (1996); Sitnickaet al., Blood, 87:4998-5005 (1996). In vivo studies have confirmed theseconclusions. Kimura et al., Proc. Natl. Acad. Sci. U.S.A., 95:1195-1200(1998). The importance of TPO in stem cell self renewal and expansionwas also supported by the clinical observation that mutations of thec-Mpl gene caused congenital amegakaryocytic thrombocytopenia, a diseasein which all hematopoietic lineages fail during childhood. Ballmaier etal., Blood, 97:139-146 (2001). It has been found that expansion of HSCsin adult bone marrow is 10 to 20 times less robust in tpo−/− micefollowing bone marrow transplantation. Exogenously added TPO rescuedthis defect. Fox et al., J. Clin. Invest., 110:389-394 (2002). Thesereports indicate that TPO is a major non redundant contributor to selfrenewal and expansion of HSCs.

Autologous stem cell transplantation (ASCT) is increasingly widely usedas a means of reconstituting the bone marrow following theadministration of potentially curative, myeloablative, high dosechemotherapy. The basis for this technique is to mobilize HSCs from bonemarrow to peripheral blood (using G-CSF+/−priming chemotherapy) fromwhich they are harvested by apheresis. These stem cells, which form aminority of the harvested population, are then capable of reconstitutingthe bone marrow when reinfused following myeloablative therapy. Stemcells obtained from peripheral blood in this technique appear to besimilar to cord blood cells and superior to bone marrow cells in theirability to regenerate bone marrow following myeloablative therapy withtime to neutrophil and platelet engraftment of less than 10 days. Themost common tumor types in which ASCT is used are myeloma, lymphoma(both Hodgkins Disease and Non-Hodgkins Lymphoma) and Acute MyeloidLeukemia. High dose chemotherapy with ASCT may be increasingly used asfirst line therapy, particularly in myeloma, but it is also used assalvage therapy following the failure of first line chemotherapy. Suchsubjects often have been heavily pretreated and thus have bone marrowwith impaired hematopoietic potential.

Following the re-infusion of these harvested cells into the subject,there is a period of time during which the subject, e.g., a humanpatient, is at risk of infection (low neutrophils) and bleeding (lowplatelets). This period of time varies depending on the number ofre-infused stem cells, which in turn depends on the ability to stimulatethe expansion of stem cells from bone marrow. Further, some subjectsalso develop bone marrow failure after an initial period of engraftment.

Stem cell transplantation is also used in an allogeneic setting whenperipheral blood stem cells are mobilized and harvested from HLA matcheddonors. Such allogeneic transplants are less frequently employed thanASCT because of the incidence of graft versus host disease but may beused when it is not possible to obtain sufficient stem cells from thepatient. However the use of allogeneic stem cells to obtain partialengraftment in the absence of complete myeloablation (the ‘minitransplant’) may also offer some therapeutic benefit due to a graftversus tumor effect. Another possible use, currently in an extremelysmall number of patients is in the field of gene therapy where normalallogeneic bone marrow cells or autologous cells transduced with anormal copy of a defective gene may be curative for some inheriteddisease caused by single gene defects. Allogeneic transplants are alsounder investigation as a therapeutic option for autoimmune diseases.

Despite the potential utility and simplicity of ASCT, there aresignificant limitations to its widespread use beyond the expected periodof pancytopenia, for which intensive subject support is required toallow the re-infused cells to resume levels of hematopoiesis sufficientto maintain peripheral blood counts. A significant proportion (up to40%) of transplanted subjects requires prolonged platelet transfusionsfollowing transplant (primary failure of engraftment). A smaller group(5-10% in autologous but >20% with allogeneic transplant) developsecondary thrombocytopenia despite initial engraftment, sometimesrequiring prolonged transfusions. Failure of engraftment or delayedengraftment is associated with increased mortality, increased healthcarecosts and decreased subject quality of life.

There thus exists a need to increase HSC production in such subjects.Studies have demonstrated that administration of TPO to patients resultsin mobilization of peripheral blood progenitor cells. One studydemonstrated the mobilization of colony forming cells from multiplelineages and CD34+ cells into the peripheral blood following multipledose administration of TPO in combination with G-CSF. Another studyidentified a 6 fold increase in circulating CD34+ cells 3-7 days afteradministration of a single dose of TPO in cancer patients with otherwisenormal hematopoiesis. In this study, a stem cell enriched subfraction(CD34+Thy+Lin−) was increased nearly 9 fold and the committedmegakaryocytic subfraction (CD34+CD41+CD14−) was increased nearly 15fold. This study suggests that TPO is capable of mobilizing bothself-renewing HSC and committed daughter cells from bone marrow.Although the availability of recombinant TPO (rhTPO) has shown promisein increasing HSC production, a need exists for an improvement in TPOtherapy by way of the mode of drug delivery

There thus exists a need for small molecule mimetic compounds of TPOthat retain substantially the full agonist activity of TPO, while at thesame time permitting various modes of administration.

There also exists a need for small molecule mimetic compounds of TPOhaving reduced immunogenicity relative to one or more of rhTPO andrhIL-11 as well as improved pharmacokinetic profile relative to one ormore of rhTPO and rhIL-11.

SUMMARY OF THE INVENTION

The present invention is directed to a method of increasing HSCproduction in a subject comprising a step of administering a TPO mimeticcompound to the subject. The TPO mimetic compound may be administered tothe subject alone or in a pharmaceutically acceptable carrier. The TPOmimetic compound can be employed alone or can be combined with one ormore additional TPO mimetic compounds and/or other agents that canenhance stem cell mobilization from bone marrow, including, e.g., G-CSF,SCF, IL-3 and/or Flt-3.

The present invention is thus also directed to a TPO mimeticpharmaceutical composition that comprises an effective amount of a TPOmimetic compound and a pharmaceutically acceptable carrier. An effectiveamount of a TPO mimetic compound is present when upon administration theTPO mimetic compound enhances expansion of the stem cell populationwithin bone marrow of a subject and/or mobilizes the stem cells into theperipheral circulation of a subject.

The present invention is also directed to a method of providing HSCs toa subject. The method can include the steps of administering a TPOmimetic compound to the subject to enhance expansion of the stem cellpopulation within bone marrow and/or to mobilize the stem cells into theperipheral circulation. Next, the method can include harvesting one ormore stem cells from the subject from either the bone marrow or from theperipheral circulation and then transplanting the harvested one or morestem cells into the subject.

The present invention is also directed to a method of providing HSCsfrom a donor subject to a recipient subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic which represents regulation of platelet and HSCproduction in accordance with the method of the present invention.

FIG. 2 is a listing of compounds, which can be suitable for use in themethod of the present invention. (Left Column: SEQ ID NOS: 1-4, 1, 2,and 5-12; middle column: SEQ ID NOS: 13-17, 17, and 18-24; right column:SEQ ID NOS: 1-2, 25-30, 29, 31, and 32, respectively in order ofappearance).

DETAILED DESCRIPTION OF THE INVENTION

The relevant portions of the patent publications and literature citedherein are incorporated by reference herein.

In one embodiment, the present invention is directed to increasing HSCproduction by administering to a subject a TPO peptide, a TPO mimeticcompound, including, but not limited to the compounds set forth in FIG.2, and PEGylated forms of the compounds set forth in FIG. 2. Themethodology that may be employed for PEGylation of the compounds setforth in FIG. 2 is described in U.S. Pat. No. 5,869,451.

In an embodiment, the present invention is directed to increasing HSCproduction by administering to a subject a TPO peptide, as described incorresponding U.S. application Ser. No. 60/498,740 (attorney docket no.038073-5005 PR), filed Aug. 28, 2003, the entire contents of which areincorporated herein by reference. According to this embodiment, the TPOpeptide is a compound having (1) a molecular weight of less than about5000 daltons, and (2) a binding affinity to TPO receptor as expressed byan IC₅₀ of no more than about 100 μM, wherein from zero to all of the—C(O)NH-linkages of the peptides have been replaced by a linkageselected from the group consisting of —CH₂OC(O)NR-linkage; a phosphonatelinkage; a —CH₂S(O)₂NR-linkage; a CH₂NR-linkage; a C(O)NR⁶ linkage; anda —NHC(O)NH-linkage where R is hydrogen or lower alkyl and R⁶ is loweralkyl, further wherein the N-terminus of said compound is selected fromthe group consisting of a —NRR¹ group; a —NRC(O)OR group; a —NRS(O)₂Rgroup; a —NHC(O)NHR group; a succinimide group; a benzyloxylcarbonyl-NHgroup; and a benzyloxycarbonyl-NH group having from 1 to 3 substituentson the phenyl ring selected from the group consisting of lower alkyl,lower alkoxy, chloro and bromo, where R and R¹ are independentlyselected from the group consisting of hydrogen and lower alkyl, andstill further when the C-terminus of the compound has the formula—C(O)R² where R² is selected from the group consisting of hydroxy, loweralkoxy, and —NR³R⁴ where R³ and R⁴ are independently selected from thegroup consisting of hydrogen and lower alkyl and where the nitrogen atomof the —NR³R⁴ group can optionally be the amine group of the N-terminusof the peptide so as to form a cyclic peptide, and physiologicallyacceptable salts thereof.

In a related embodiment, the TPO mimetic peptide comprises a sequence ofamino acids X₉X₈GX₁X₂X₃X₄X₅X₆X₇, where X₉ is A, C, E, G, I, L, M, P, R,Q, S, T or V; and X₈ is A, C, D, E, K, L, Q, R, S, T or V; and X₆ is aβ-(2-napthyl)alanine (referred to herein as “2-Nal”) residue. Morepreferably, X₉ is A or I, and X₈ is D, E or K. Further, X₁ is C, L, M,P, Q or V; X₂ is F, K, L, N, Q, R, S, T or V; X₃ is C, F, I, L, M, R, S,V or W; X₄ is any of the 20 genetically coded L-amino acids; X₅ is A, D,E, G, K, M, Q, R, S, T, V or Y; and X₇ is C, G, I, K, L, M, N, R or V.

A particularly preferred TPO mimetic peptide is I E G P T L R Q (2-Nal)L A A R A (SEQ ID NO: 33).

Another particularly preferred TPO mimetic peptide is I E G P T L R Q(2-Nal) L A A R X₁₀, wherein X₁₀ is a sarcosine or β-alanine residue ora pegylated form of this compound.

In another embodiment, the TPO mimetic peptide is dimerized oroligomerized to increase the affinity and/or activity of the compound.An example of such a compound includes (SEQ ID NO: 34):

where X₁₀ is a sarcosine or β-alanine residue or a pegylated form ofthis compound. The pegylated form may include a 20 k MPEG residuecovalently linked to each N-terminal isoleucine.

According to another embodiment, the TPO mimetic peptide has thefollowing formula:

where X₁₀ is a sarcosine or β-alanine residue or a pegylated form ofthis compound. This structure can also be represented by the followingstructure (H-I E G P T L R Q (2-Nal) L A A R X₁₀)₂K—NH₂. The pegylatedform may include a 20 k MPEG residue covalently linked to eachN-terminal isoleucine.

One or more TPO mimetic peptides, and in particular PEGylated TPOmimetic peptides (collectively referred to herein as “TPO mimeticcompounds” or “TPO mimetic compounds of the invention”), can be used toincrease the number of stem cells in bone marrow. Important datasupporting the use of a TPO mimetic compound in ASCT is provided by astudy performed by Somlo et al., Blood, 93(9):2798-2806 (1999), in whichrecombinant human thrombopoietin (rhTPO) was able to enhance themobilization and apheresis yields of CD34+ stem cells in response toG-CSF with consequent reduction in the number of aphereses. Subsequentlythe engraftment of reinfused cells was also improved in terms of reducedtime to ANC>0.5×10⁹/L and platelet transfusion independence, though thiseffect did not reach statistical significance in the small sample sizeused in this pilot study. By increasing the number of stem cells, thetotal harvest of stem cells from the subject can be significantlyimproved. Further, by increasing the number of stem cells harvested fromthe subject, the number of stem cells available for transplantation backinto the subject can also be significantly improved, thereby potentiallyreducing the time to engraftment (the time during which the subject hasinsufficient neutrophils and platelets), thus preventing complications.

In addition, the present invention can also reduce the proportion ofsubjects who are unable to harvest enough cells to proceed withtreatment for their primary illness, e.g., chemotherapy and other bonemarrow ablative treatments. Furthermore, the proportion of the number ofsubjects with delayed primary engraftment can also be reduced.

TPO mimetic compounds such as those in FIG. 2 and disclosed herein canbe used to increase HSC production. This is accomplished byadministering one or more of the compounds to a subject. The compoundsset forth in FIG. 2 and disclosed herein, as well as PEGylated forms ofthe compounds, set forth in FIG. 2 can have reduced immunogenicityrelative to one or more of rhTPO and rhIL-11 and can also have animproved pharmacokinetic profile relative to one or more of rhTPO andrhIL-11.

TPO mimetic compounds can also be used to provide autologous HSCs to asubject. Typically, this involves the steps of administering a TPOmimetic compound to a subject in need thereof to enhance expansion ofthe stem cell population within bone marrow and/or to mobilize the stemcells in peripheral circulation; harvesting one or more of the bonemarrow stem cells or one or more of the stem cells in the peripheralcirculation; and transplanting the one or more harvested stem cells backinto the subject.

In addition, the stem cells obtained from harvesting according to methodof the present invention described above can be cryopreserved usingtechniques known in the art for stem cell cryopreservation. Accordingly,using cryopreservation, the stem cells can be maintained such that onceit is determined that a subject is in need of stem cell transplantation,the stem cells can be thawed and transplanted back into the subject.

The TPO mimetic compounds, including the compounds set forth in FIG. 2and disclosed herein as well as the PEGylated forms of the compounds setforth in FIG. 2, can thus be used for, inter alia: reducing the time toengraftment following reinfusion of stem cells in a subject; reducingthe incidence of delayed primary engraftment; reducing the incidence ofsecondary failure of platelet production; and reducing the time ofplatelet and/or neutrophil engraftment following reinfusion of stemcells in a subject. These methods typically include the steps ofadministering a TPO mimetic compound to a subject in need thereof toenhance expansion of the stem cell population within bone marrow and/ormobilize the stem cells in peripheral circulation and then harvestingone or more of the bone marrow stem cells or the stem cells in theperipheral circulation and then transplanting the harvested stem cellback into the subject at the appropriate time, as determined by theparticular needs of the subject.

The method of the invention may also be used to increase the number ofstem cells from a donor subject whose cells are then used for rescue ofa recipient subject who has received bone marrow ablating chemotherapy.

A. Dosage Forms and Routes of Administration

The TPO mimetic compounds useful for the present invention can beadministered as pharmaceutical compositions comprising, as an activeingredient, at least one of the peptides or peptide mimetics set forthin FIG. 2 and/or disclosed herein and/or described in U.S. Pat. No.5,869,451, the entire content of which is hereby incorporated byreference, in association with a pharmaceutical carrier or diluent. Thecompounds can be administered by oral, pulmonary, parental(intramuscular, intraperitoneal, intravenous (IV) or subcutaneousinjection), inhalation (via a fine powder formulation), transdermal,nasal, vaginal, rectal, or sublingual routes of administration and canbe formulated in dosage forms appropriate for each route ofadministration. See, e.g., Bernstein, et al., PCT Patent Publication No.WO 93/25221; Pitt, et al., PCT Patent Publication No. WO 94/17784; andPitt, et al., European Patent Application 613,683, each of which isincorporated herein by reference.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound can be admixed with at least one inert pharmaceuticallyacceptable carrier such as sucrose, lactose, or starch. Such dosageforms can also comprise, as is normal practice, additional substancesother than inert diluents, e.g., lubricating agents such as magnesiumstearate. In the case of capsules, tablets, and pills, the dosage formsmay also comprise buffering agents. Tablets and pills can additionallybe prepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, with the elixirscontaining inert diluents commonly used in the art, such as water.Besides such inert diluents, compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring, and perfuming agents.

Preparations for parental administration include sterile aqueous ornon-aqueous solutions, suspensions, or emulsions. Examples ofnon-aqueous solvents or vehicles are propylene glycol, polyethyleneglycol, vegetable oils, such as olive oil and corn oil, gelatin, andinjectable organic esters such as ethyl oleate. Such dosage forms mayalso contain adjuvants such as preserving, wetting, emulsifying, anddispersing agents. They may be sterilized by, for example, filtrationthrough a bacteria retaining filter, by incorporating sterilizing agentsinto the compositions, by irradiating the compositions, or by heatingthe compositions. They can also be manufactured using sterile water, orsome other sterile injectable medium, immediately before use.

Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance,excipients such as cocoa butter or a suppository wax. Compositions fornasal or sublingual administration are also prepared with standardexcipients well known in the art.

The compositions of the invention can also be microencapsulated by, forexample, the method of Tice and Bibi (in Treatise on Controlled DrugDelivery, ed. A. Kydonieus, Marcel Dekker, New York (1992), pp.315-339).

The composition can also be combined with, inter alia, G-CSF, SCF, IL-3or Flt-3 and/or other agents that can enhance stem cell mobilizationfrom bone marrow (including priming chemotherapy and integrinantagonists).

B. Dosage Amount

The quantities of a TPO mimetic compound necessary for the presentinvention will depend upon many different factors, including means ofadministration, target site, physiological state of the subject, andother medicants administered. Thus, treatment dosages should be titratedto optimize safety and efficacy. Typically, dosages used in vitro mayprovide useful guidance in the amounts useful for in situ administrationof these reagents. Animal testing of effective doses for treatment ofparticular disorders will provide further predictive indication of humandosage. Various considerations are described, e.g., in Gilman, et al.(eds), Goodman and Gilman's: The Pharmacological Basis of Therapeutics,8th ed., Pergamon Press (1990); and Remington's Pharmaceutical Sciences,7th Ed., Mack Publishing Co., Easton, Pa. (1985); each of which ishereby incorporated by reference.

The TPO mimetic compounds are useful for the present invention whenadministered at a dosage range of from about 0.001 mg to about 20 mg/kgof body weight per day. Alternatively, in some instances 0.0001 mg/kg toabout 10 mg/kg may also be administered. The specific dose employed isregulated by the particular condition being treated, the route ofadministration as well as by the judgement of the attending cliniciandepending upon factors such as the severity of the condition, the ageand general condition of the subject, and the like.

C. Subjects and Indications

As used herein, a subject includes anyone who is a candidate forautologous stem cell or bone marrow transplantation during the course oftreatment for malignant disease or as a component of gene therapy. Otherpossible candidates are subjects who donate stem cells or bone marrow tosubjects for allogeneic transplantation for malignant disease or genetherapy.

In order to provide an acceptable probability of engraftment, a minimumnumber of stem cells must be harvested. Though not precisely defined, itis generally accepted that 2-3×10⁶ CD34⁺ cells per kg must be harvestedin order to provide a reasonable chance of engraftment. Reinfusion of5×10⁶/kg cells appears to produce optimum results in terms of time toengraftment. This large number of cells is required because the actualnumber of the specific subset of CD34⁺ cells that are capable of longterm reconstitution of bone marrow is very small. As many as 20% oftransplanted patients are considered to be poor mobilizers, requiringmultiple aphereses to generate sufficient cells. Although one of themost important predictors of poor stem cell mobilization is the age ofthe patient, heavy pretreatment with chemotherapy is also a significantfactor. There are likely a large number of patients, particularlyelderly patients with myeloma or NHL, who are not considered for ASCTbecause of the low probability of successful engraftment.

Consequently the method of the invention provides a solution to theunmet need in ASCT, i.e., the need to improve the proportion of patientswho have successful and rapid engraftment. This is achieved primarilydue to improvement in mobilization of stem cells, either by increasingnumbers of mobilized cells or by increasing the proportion of HSCs inthe mobilized CD34+ population. The method of the invention thusprovides the following benefits:

-   -   1. Allows transplantation to proceed in patients who would not        otherwise be considered as candidates because of the        unacceptably high risk of failed engraftment;    -   2. Reduces the number of aphereses required to generate a        minimum acceptable harvest;    -   3. Reduces the incidence of primary and secondary failure of        engraftment by increasing the number HSCs available for        transplantation; and    -   4. Reduces the time required for primary engraftment by        increasing the number of committed precursors of the important        hemopoietic lineages.

In accordance with the established effect of TPO on HSCs, the TPOmimetic compounds of the invention may have the following clinicalbenefits in stem cell transplantation:

-   -   Improvement of apheresis yields: Numerous studies have suggested        that the number of reinfused CD34⁺ stem cells is an important        factor in determining the time to engraftment. As demonstrated        with TPO, the addition of a TPO mimetic compound may increase        mobilization of CD34⁺ cells as an adjunct to conventional        mobilization regimens of G-CSF and chemotherapy. The primary        benefit would be to improve the prospect for rapid and        subsequent long term engraftment. Reducing the number of        aphereses required to generate an acceptable number of cells        would reduce cost and patient inconvenience. Improvement in        apheresis yields would be of particular benefit in patients with        risk factors for low mobilization (age and heavy pretreatment).        Such patients may otherwise not be candidates for ASCT.    -   Improvement of the engraftment potential of apheresed cells:        Long term engraftment following myeloablative therapy is        produced by a small fraction of the CD34⁺ cell population (most        likely within the CD34⁺CD38⁻Lin⁻ population). Because they are        so rare, large numbers of CD34⁺ cells are required to provide        effective engraftment (2-5×10⁶/kg). G-CSF does not affect the        proportions of different subtypes of CD34⁺ cells and is simply        used as an agent that can increase the number of these cells in        peripheral blood prior to harvest. Priming chemotherapy may        actually be toxic to such cells. However TPO is increasingly        widely recognized as an agent that can increase the self-renewal        of the most primitive stem cells, and thus capable of long-term        hemopoietic reconstitution. A similar effect of a TPO mimetic        compound may therefore increase the proportion of the CD34⁺ cell        population that can contribute to long-term engraftment and thus        reduce the risk of failure of engraftment. TPO may also increase        the numbers of stem cells committed to the megakaryocytic        lineage thus producing earlier independence from platelet        transfusions, i.e., reduced time to engraftment.

The two beneficial effects described above may be additive orsynergistic, leading to greater reduction in time to engraftment thanmight be seen with agents that only increase mobilization of stem cells.

The use of a TPO mimetic compound of the invention would likely requireonly a small number of doses given, e.g., intravenously orsubcutaneously, prior to apheresis. Such a dosing regimen would minimizethe risk of significant antigenicity, which is already predicted to below due to the use of a pegylated product.

The TPO mimetic compound of the invention is first administered tonormal volunteers:

-   -   1. To establish the effect of the TPO mimetic compound on        peripheral blood CD34⁺ cell populations, platelet counts and        other hematological parameters;    -   2. To establish the preliminary safety profile of the TPO        mimetic compound in terms of dose limiting toxicity and high        frequency adverse events;    -   3. To determine the most appropriate dose, dose regimen and dose        timing of pre-apheresis dosing with the TPO mimetic compound;        and    -   4. To determine the pharmacokinetic profile of the TPO mimetic        compound in humans.    -   5. To generate preliminary comparative information on the        effects of a TPO mimetic compound of the invention and G-CSF on        the multilineage potential of peripheral blood stem cells.

Normal human volunteers, which are the most appropriate population forevaluation of pharmacokinetics and initial safety profile, provide theclearest understanding of the effects of the TPO mimetic compound onHSCs because of the absence of the background effects of chemotherapyand disease.

The study will be a single blind, dose rising study in which normalhuman volunteers receive a single intravenous dose of the TPO mimeticcompound of the invention, given as a one hour infusion. The startingdose will be 15 ug/kg. Successive dose cohorts will receive 25, 50, 100and 200 ug/kg. Four subjects will be enrolled in each cohort, three ofwhom will receive active therapy and one whom will receive placebo. Eachsubject will be observed at regular (15 minute) intervals during theinfusion and will remain as an inpatient for 24 hours for close safetymonitoring and pharmacokinetic sampling. Further outpatient follow upfor safety, pharmacokinetic and pharmacodynamic evaluation will occur ondays 2, 4, 7, 14, 21 and 28. Each successive dose cohort will be treatedtwo weeks after the previous group.

When a dose is reached at which evidence of pharmacodynamic effect(defined as a 50% increase in platelet count relative to thepretreatment value) is observed in 2/3 of the actively treated subjectsdosing at that level will be extended to include a further four subjects(3/1 active/placebo). If the efficacy is confirmed, one further dosecohort of six subjects (4/2 active/placebo) will be enrolled to providefurther confirmation of the pharmacodynamic effect. If thepharmacodynamic effect is only observed at the highest planned dose, afurther dose increase will be considered, assuming that no evidence oftoxicity has been observed.

If a safety/tolerability event that is possibly or probably related tostudy medication occurs in a single actively treated subject at any dosefour further subjects (3/1 active/placebo) will be enrolled at that doseto determine if a dose limiting toxicity has been identified.

Blood samples will be taken for measurement of drug levels 30 minutesafter the beginning of the infusion, at the end of the infusion and atthe following times after the end of the infusion: 5 minutes, 15minutes, 30 minutes, 1, 2, 4, 8, 12, 24, 48, 96 hours and 7 and 14, 21and 28 days. Compound levels will be measured using either a cell basedbioassay or by ELISA.

For comparison, a single dose of G-CSF will be administered to threesubjects to measure the effects on CD34+ cells.

The effect of the TPO mimetic compound on peripheral blood CD34⁺ counts,if any will be delayed for several days (3-7 if the effect is similar tothrombopoietin). Furthermore because of the unknown impact of the TPOmimetic compound pharmacokinetics on the PD profile it is not certainwhen the maximal effect of a single dose will be seen. It is the timingof the maximal effect that will determine the interval between the TPOmimetic compound dosing and harvest of CD34⁺ cells in subsequent patientstudies. A good correlation between peripheral blood CD34⁺ counts andthe yield in subsequent harvesting has been demonstrated suggesting thatthis approach is reasonable. It may be appropriate to ensure that theTPO mimetic compound invention is given prior to G-CSF to enable anexpansion of the HSC population within bone marrow followed bymobilization of the expanded population. Most studies that use G-CSF tostimulate mobilization give the drug for five days with the harveststated towards the end of the dosing period. It is possible that thepharmacodynamic profile of the TPO mimetic compound will require it tobe dosed some days prior to G-CSF.

The impact of the TPO mimetic compound on the number of self-renewingHSCs in the mobilized population could provide an increase in selfrenewal capacity which could lead to successful engraftment with lowernumbers of mobilized cells, greater ease of performing tandemtransplants and the possibility that the TPO mimetic compound couldeventually replace G-CSF as the standard mobilizing agent.

This aspect of the clinical pharmacology of the TPO mimetic compound canbe addressed by measuring the self renewal capacity of CD34⁺ populationsfrom the normal volunteers study, both in in vitro studies of theability to sustain long term colony formation (the LTC-IC culture) andby performing SCID/NOD mouse repopulation assays in which the mobilizedcells are infused into lethally irradiated SCID/NOD mice. Preliminarycalculations indicate that performing such studies should be feasiblewith the CD34⁺ cells that are contained in 30-50 mls of blood, providedthat the CD34⁺ count has risen to approximately 15×10³/ml.

The assumptions underlying this statement are outlined below:

-   -   1. PBMCs from normal subjects will be obtained by Ficoll/Hypaque        separation and then Lin+ cells will be removed by negative        selection. CD34+CD38− subfractions of this enriched population        will then be isolated by FACS and administered to SCID/NOD mice.        Mice will also receive accessory cells and growth factors to        permit the use of lower numbers of CD34+CD38−Lin− cells per        mouse (Bonnet et al., Bone, Marrow Transplantation, 23:203-209        (1999)). Alternatively, the original PBMC population will be        used without further purification to provide both repopulating        and accessory cells.    -   2. The primary endpoint for this assay will be survival of the        recipient mice. However Southern Blot analyses will also be        performed to detect human DNA in the recipient mice. If        possible, detection of human progenitor cells will be determined        by human selective long term marrow cultures and/or flow        cytometry with human specific MAbs.    -   3. Each subject will provide enough blood to test four        CD34+CD38−Lin− cell doses (250, 500, 1000 and 2000 cells/mouse).        Each cell dose will be given to 5 mice. With these design        assumptions approximately 1.9×10⁴ CD34+CD38−Lin− cells will be        needed from each subject. If additional in vitro colony forming        studies are performed, more cells will be needed.    -   4. Each normal subject will provide this blood sample only once        and only when the CD34+ cell count in peripheral blood has        reached 15×103/ml. CD34+CD38−Lin− population represents 5-8% of        CD34+ population (Gallacher et al., Blood, 95:2813-2820 (2000)).        Studies with TPO in normal volunteers indicated that 16×10³        CD34+ cells/ml were seen in the peripheral blood.    -   5. 30 mls of blood will be required from each subject to yield        2.25-3.6×104 cells.    -   6. It will not be possible to perform these studies in placebo        treated subjects due to low levels of CD34+ cells (<3×10³/ml).        For comparison, similar quantities of cells will be taken from        subjects treated with G-CSF. Equal numbers of cells will be        infused into the mice.    -   7. The validity of these assumptions is tested with independent        data. Approximately 1 in 6×10⁶ PBMCs is capable of repopulating        a SCID/NOD mouse (Wang et al., Blood, 89:3919-3924 (1997)). Of        this population, the CD34+ population is 0.13-0.39% and 5-8% of        this subset is CD34+CD38−Lin− (Tichelli et al., Br. J. Hematol.,        106:152-158 (1999)). This represents 390-1872 cells from the        original 6×10⁶ PBMCs. In a separate study the incidence of        SCID/NOD repopulating cells in the CD34+CD38−Lin− population has        been demonstrated to be 1 in 617 (Bhatia et al., PNAS,        94:5320-5325 (1997)). This number is consistent with the        extrapolation to the incidence in unselected cells.    -   8. If cell number becomes limiting, the highest cell dose cohort        of mice will be dropped.

CD34⁺ cells taken from volunteers who are given G-CSF will be used as acontrol for these studies.

The proposed normal volunteer study will provide the database requiredto determine the design of patient studies in terms of dose, doseregimen and dose timing as well as strong pharmacodynamic evidence thatpredicts clinical efficacy. The next phase of the clinical pharmacologyprogram will seek to reproduce the observed effects in patientsscheduled for stem cell transplantation as well as providingtranslational data that will demonstrate a link between thepharmacodynamic endpoints described above and the clinical endpointsrequired for regulatory approval. The mimetic compound of the presentinvention is then administered to patients in need:

-   -   1. To explore the risk to benefit profile of the TPO mimetic        compound in different populations of patients who are candidates        for autologous stem cell transplantation; and    -   2. To obtain preliminary evidence of the likely effect of the        TPO mimetic compound on apheresis yields of peripheral blood        CD34⁺ stem cells and post engraftment outcomes.

The first patient study will again be a single dose, dose rising design(assuming that there is no reason to give the dose of the TPO mimeticcompound as a divided dose). Dosing of the TPO mimetic compound will beintroduced into a standard mobilization regimen, with the dose intervalbetween dosing and harvest predicted from the volunteer study. The samepharmacodynamic endpoints will be evaluated in this study as in theprevious study, but data on apheresis yields, number of aphereses andsubsequent rates and times of engraftment will also be obtained. Dosingwill be given via single use 10-20 mg vial containing lyophilized powderas a single dose by intravenous bolus administration before apheresisand after reinfusion of harvested cells. A subcutaneous dosingbioequivalent can be administered with intravenous dosing. It isexpected that the dose would be between about 10-300 μg/kg.

A key aspect of this study will be to explore the risk to benefit of theTPO mimetic compound in different patient populations. An increasingnumber of patients are receiving high dose, myeloablative therapy withASCT relatively early in the course of their disease. Such patientsoften have relatively normal bone marrow and, particularly if they areyoung, are likely to mobilize acceptable numbers of CD34+ cells withconsequent high likelihood of rapid engraftment. In this population, thepotential impact of an additional agent to enhance mobilization may belimited but could be manifest as even more rapid engraftment withreservation of harvested cells for tandem transplant. Nevertheless, thispopulation, which most closely resembles the normal population at leastin terms of bone marrow responsiveness, is an important translationalgroup for the development of the TPO mimetic compound.

Patients who become candidates for ASCT after multiple previous coursesof therapy often have greater difficulty in generating enough CD34⁺cells for an adequate harvest. Consequently, many of these patientsrequire prolonged apheresis schedules and a higher incidence of delayedor failed engraftment. A proportion of these patients are not able toundergo autologous transplant and must instead resort to allogeneictransplant with increased risk of post transplant complications. It isthis population in which an additional mobilization agent may be ofgreat benefit.

Consequently, the first patient study will enroll patients from bothcategories. The data from the ‘good mobilizers’ will be used as abenchmark to determine the impact of the TPO mimetic compound on the‘poor mobilizers’. A non-treated group, receiving only standard of carewill be included.

The pharmacodynamic endpoints outlined above will provide a robustsurrogate of the likely clinical benefit of the TPO mimetic compound inASCT.

Definitive studies will be conducted as parallel group, double blind,placebo controlled studies. Once randomization has occurred clinicaldecisions about transplantation will be made according to predefinedrules and accepted clinical practice.

The primary endpoint for the studies will be mean time to engraftmentfollowing re-infusion of harvested cells. Time to engraftment will bedefined as number of days until platelet count is maintained above20×10⁹/L without transfusion support for a period of 7 days.

Secondary endpoints will include:

-   -   1. Time to neutrophil engraftment (defined as neutrophil count        maintained above 0.5×10⁹/L);    -   2. Time to platelet count >50×10⁹/L (maintained for 7 days        without transfusion support);    -   3. Proportion of patients with delayed platelet engraftment;    -   4. Proportion of patients with secondary failure of platelet        engraftment;    -   5. Proportion of patients who fail to generate minimum harvest        necessary for transplantation;    -   6. CD34⁺ harvest (CD34⁺ cells/kg);    -   7. Number of aphereses required for harvest; and    -   8. Number of platelet transfusions.

A key factor in study design will be selection of the target population.Published data indicates that the number of CD34⁺ cells harvested is amajor determinant of subsequent engraftment kinetics and will thereforedirectly impact the primary endpoint of the studies. The key demographicfeatures that will influence the ability to mobilize CD34⁺ cells is theamount of pretreatment and patient age. A number of issues must beconsidered:

-   -   1. If a poor mobilizing population is selected there will be the        greatest opportunity to detect an improvement in engraftment        rates but the ability of the bone marrow to respond to the TPO        mimetic compound may be so compromised that no response is        possible;    -   2. If a high mobilizing population is selected the ability to        detect a response over background therapy may be limited due to        the fact that optimal numbers of self renewing HSCs will be        re-infused regardless of the addition of the TPO mimetic        compound;    -   3. The intrinsic effect of the TPO mimetic compound of the        invention to increase mobilization may prevent accurate        definition of good or poor mobilizers;    -   4. The ability to detect the effect on engraftment of an        increase in self renewing HSCs may only be seen in patients in        whom the number of these cells is a limiting factor in        engraftment kinetics.

On the basis of these issues it is important that the study populationfor these studies is limited in the number of patients at the extremesof the mobilization range. At either extreme it may be difficult todemonstrate the efficacy of the compound. This can be achieved byexcluding some patient groups that are highly likely to contribute toextreme values of mobilization (for example patients receiving firstline therapy, patients with myelodysplasia and/or low marrow reserve)and also by ensuring that the sample size is determined by patients whoreach a predefined range of CD34⁺ harvest size (i.e., randomizedpatients who failed to meet these criteria would be replaced).

If a design of this type is followed, the majority of the patients whowould contribute to the primary endpoint in the placebo group would haveCD34⁺ yield falling into the following categories in the ratio 2:3:1respectively:

<2.0×10⁶/kg (median time to engraftment=17 days)

2-5×10⁶/kg (median time to engraftment=12 days)

>5×10⁶/kg. (median time to engraftment=10 days).

In a population of this type, the expected median time to engraftmentwould be 13-14 days. If the effect of the TPO mimetic compound on yieldof CD34⁺ cells was to alter the proportions of the different harvestcategories from 2:3:1 to 1:2:3, this change alone would result in areduction in the median time to engraftment of 1.66 days. If an improvedtime to engraftment, within each category caused by increased numbers ofself-renewing HSCs, is superimposed on this such that the median time toengraftment improves by 5 days in the lowest yield group (i.e., theybehave like the middle yield group) and 2 days in the middle group(i.e., they behave like the high yield group), the additional reductionin median time to engraftment would be 1.66 days. No effect on time toengraftment is assumed for the high yield group. Collectively, theimpact of the TPO mimetic compound treatment on median time to plateletengraftment, for the purposes of calculating a sample size, would be 3days. To enable the maximum opportunity to define the clinical benefitof the TPO mimetic compound a relatively low threshold for the minimumharvest required to allow myeloablation to proceed should be set.

The ability to demonstrate efficacy of the TPO mimetic compound in ASCTis relatively straightforward because the observation of increasednumbers of CD34⁺ stem cell in the peripheral blood of normal volunteerstreated with single doses will suffice. The first human study willtherefore demonstrate a biologically relevant effect. Several studieshave identified the level of CD34⁺ cells in peripheral blood as animportant predictor of subsequent apheresis yield. However the effect onstem cell mobilization in combination with G-CSF will not be establisheduntil the first patient study is completed. It will be more difficult toestablish that the mobilized CD34⁺ cells contain increased numbers ofstem cells, in part because it is difficult to measure the low levels ofHSCs in unstimulated patients. However since G-CSF is reported not toaffect the proportion of HSCs in the CD34⁺ population, it may bepossible to infer some effect of the TPO mimetic compound on the numberof self renewing HSCs within the mobilized cell population by comparisonto cells mobilized with G-CSF.

The most important predictor of success will be apheresis yield. Thenumber of reinfused cells is an important predictor of the subsequenttime to engraftment. Consequently the proportion of patients withclinically acceptable or high yields will be a major determinant of thelikely impact on time to engraftment and the proportion of patients withdelayed or failed engraftment.

It is expected that the TPO mimetic compound is as good as or superiorto G-CSF in mobilizing stem cells and that the TPO mimetic compoundprovides improved quality of the mobilized stem cell population.

A single blind study to evaluate the effect of the TPO mimetic compoundon mobilization of peripheral blood CD34+ stem cells when added tostandard mobilization regimens in patients scheduled for myeloablativechemotherapy with autologous stem cell transplantation.

To determine the effect of the TPO mimetic compound on the mobilizationof stem cells prior to apheresis, a single dose, dose rising study usingdoses proven to mobilize CD34+ cells in normal volunteers will beconducted. Each dose cohort will contain six patients receiving activemedications and two receiving G-CSF background therapy only. Each cohortwill be divided into two groups of 3 active and 1 placebo patient. Onegroup will be patients receiving autologous SCT as first line therapywhereas the other will be heavily pretreated patients receivingautologous SCT as salvage therapy. When a dose is reached that producesan increased yield of CD34+ cells relative the placebo patient (effectsize to be defined) and to historical controls for the effect of G-CSFon stem cell mobilization, eight additional patients (per subcohort)will be recruited at that dose to solidify the evidence of efficacy andto explore additional pre and post transplant endpoints (to includenumber of aphereses required to yield 3×10⁶ cells/kg, the proportion ofpatients who attain an adequate harvest and the time to posttransplantation neutrophil recovery and platelet transfusionindependence). Further dose increases will continue as per the originalrandomization schedule. If one sub-cohort reaches an efficacy plateau ordose limiting toxicity, the remaining sub-cohort will continue in doseescalation. At the time of apheresis, a sample of apheresed cells willbe obtained for study of the multipotential capacity of the harvestedcells (assuming that the size of the harvest is not limiting). Aftermeeting screening criteria and collection of baseline blood samples, thepatient will be receive a single dose of study medication given byintravenous infusion over a period of 60 minutes. Follow up visits willoccur every 48 hours until stem cell harvest is complete. Stem cellharvest will be deemed to have failed if 10 aphereses have failed toyield sufficient cells for successful engraftment (minimum 2×10⁶/kg).The patient will then continue with myeloablative chemotherapy,reinfusion of stem cells, and follow up with appropriate supportive careaccording to the protocol defined for the patient's tumor. Data onengraftment will be abstracted from the source documents according topredefined specifications.

Samples will be taken for pharmacokinetic sampling at each study visit.Compound levels will be measured using an ELISA.

It is believed that the administration of the TPO mimetic compound inaccordance with the method of the invention will provide a number ofadvantages, including, inter alia:

-   -   Reduction in median time to platelet engraftment (defined as        platelet count >20×10⁹/L) of 3 days when added to standard        therapy. Reduction is 1 day when used instead of standard        therapy.    -   Reduction in the proportion of patients with delayed time to        platelet engraftment from 40% to 10%.    -   Increase in the proportion of patients who attain primary        platelet recovery (defined as patients who maintain a platelet        count >50,000 for 7 days) from 60% to 85%.    -   Reduction in number of platelet transfusions required (from a        median of 5 to a median of 3).    -   Reduction in median time to ANC>0.5×10⁹/L of 1 day.    -   Reduction in the proportion of patients who fail to meet minimum        stem cell harvests (3×10⁶/kg) for transplantation (from 35% to        5%) when used in combination with G-CSF.    -   Increase in the yield of CD34+ cells when used in combination        with G-CSF (4×10⁶/kg vs 1×10⁶/kg).    -   Reduction in the number of harvests required to yield sufficient        cells to support transplantation when used in combination with        G-CSF (from a median of 3 to a median of 1).

Convenient single dose therapy to improve the efficiency of stem celltransplantation, to permit more aggressive treatment of solid tumors,myeloma and lymphoma and to increase the number of candidates for stemcell transplantation.

Although only particular embodiments of the invention are specificallydescribed above, it will be appreciated that modifications andvariations of the invention are possible without departing from thespirit and intended scope of the invention.

1. A method of increasing hematopoietic stem cell production in asubject comprising a step of administering a TPO mimetic compound tosaid subject, wherein the TPO compound has the following sequence:

wherein (2-Nal) is β-(2-naphthyl)alanine and Sar is sarcosine.
 2. Themethod of claim 1, wherein the subject is a human.
 3. The method ofclaim 1, wherein the TPO mimetic compound has reduced immunogenicityrelative to one or more of the recombinant human thrombopoietin (rhTPO)and recombinant human interleukin-11 (rhIL-11).
 4. The method of claim 1wherein the TPO mimetic compound has an improved PK profile relative toone or more of rhTPO and rhIL-11.
 5. The method of claim 1, wherein saidTPO mimetic compound is covalently attached to a hydrophilic polymer. 6.The method of claim 5, wherein said hydrophilic polymer has an averagemolecular weight of between 500 to 40,000 daltons.
 7. The method ofclaim 6, wherein said hydrophilic polymer has an average molecularweight of between 5,000 to 20,000 daltons.
 8. The method of claim 6,wherein said hydrophilic polymer has an average molecular weight of20,000 daltons.
 9. The method of claim 5, wherein said polymer ispolyethylene glycol.